This is a competitive renewal of NIH grant DK 33201-05. During the first 4 years substantial progress has been made characterizing the kinase activity of the insulin receptor and determining its role insulin action. We have mapped the sites of autophosphorylation in the beta-subunit, demonstrated that the receptor is regulated by multisite phosphorylation, developed inhibitors of the receptor kinase, used antiphosphotyrosine antibodies to identify cellular substrates, presented evidence for conformation changes in the receptor and possible non-covalent interactions, demonstrated two pools of receptor, shown that phosphorylated insulin and EGF receptors are internalized and characterized alterations in receptor kinase activity in mutant cell lines and disease states. Using in vitro mutagenesis we also have demonstrated that although the kinase activity is required for insulin action, it may not be sufficient. In all over 60 papers are published, in press or submitted as a result of this work. During the next 5 years we propose to further our understanding of the role of tyrosine phosphorylation in insulin action in the following ways: 1. Subsets of the receptor which differ in kinase activity will be characterized and the factors accounting for the activity differences determined. 2. The fate of the phosphorylated receptor in the cell will be studied and the role of both serine and tyrosine phosphorylation as a signal for internalization will be evaluated. 3. Specific cellular substrates will be purified. Antibodies to each will be raised and the amino acid sequence and function of each molecule will be analyzed. 4. The association of the receptor with a phosphotidylinositol kinase and interaction of the receptor with G-proteins will be studied. The role of receptor conformational change in this process will be evaluated. 5. Inhibitors of the receptor kinase will be constructed and introduced into cells by physical means or by construction of expression vectors containing the desired coding sequence. 6. Using in vitro mutagenesis we will study the role of specific receptor residues in signal transduction. 7. Disease and cell culture models of altered receptor kinase activity will be further studied to determine the exact mechanism responsible for the change.